The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25°C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t1/2 value of 16 min for thermal inactivation during incubation at 40°C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly244 was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly244→Pro substitution into the enzyme. Stability studies showed that the t1/2 value for thermal inactivation of the mutant during incubation at 40°C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The kcat/Km value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.
|ジャーナル||Biochimica et Biophysica Acta - Proteins and Proteomics|
|出版ステータス||Published - 2004 1 14|
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